Biochemical and molecular characterizaton of house cricket (Acheta domesticus, Orthoptera: Gryllidae) Delta9 desaturase.

Studies of insect fatty acyl-CoA desaturases have heretofore concentrated on the Diptera and Lepidoptera. We report here the isolation and characterization of a fatty acyl-CoA Delta9 desaturase from the house cricket, Acheta domesticus (Orthoptera). Two desaturase cDNAs were isolated from a library, one of which contained two intron sequences. The clones were identical in their respective coding regions, but had divergent 5' and 3' untranslated regions. The cDNAs encode a 359 amino acid desaturase enzyme that could rescue a fatty acyl-CoA desaturase auxotrophic phenotype when expressed in yeast. Biochemical analysis of lipids from transformed yeast cells confirmed that the enzyme is a Delta9 desaturase with activity on both palmitic and stearic acid substrates. Southern blotting indicated multiple Delta9 desaturase genes within the genome. A single message that was up-regulated in fed insects vs. starved insects was observed on northern blots, indicating transcriptional regulation in response to diet.

Isolation and molecular characterization of Musca domestica delta-9 desaturase sequences.

We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two approximately 1.66 kb cDNAs were recovered. They had identical coding regions and 3' untranslated regions (UTRs), but differed in their 5' UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a delta9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more delta9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family.

Effects of developmental age, ambient temperature, and dietary alterations on delta(12) desaturase activity in the house cricket, Acheta domesticus.

Double bond formation in polyunsaturated fatty acids (PUFA) is mediated by desaturase enzymes. Certain insect species have been found to possess a Delta(12) desaturase, previously thought to occur exclusively in plants. We have begun to characterize this enzyme to determine its relatedness to those found in plants and animals. Desaturase activity can be altered significantly by a number of environmental factors in protozoa, cyanobacteria, plants, fish, and rats. We present evidence here that Delta(12) desaturase activity in Acheta domesticus is affected by developmental stage, starvation, dietary alterations, and fluctuations in ambient temperature. Highest activity is observed during the middle of the penultimate instar and 3 to 6 days after adult emergence. Starvation markedly decreases Delta(12) activity, whereas resumption of feeding on fat-free or low fat diets increases activity.

Juvenile hormone regulation of HMG-R gene expression in the bark beetle Ips paraconfusus (Coleoptera: Scolytidae): implications for male aggregation pheromone biosynthesis.

Juvenile hormone III (JH III) induces acyclic isoprenoid pheromone production in male Ips paraconfusus. A likely regulatory enzyme in this process is 3-hydroxy-e-methylglutaryl-CoA reductase (HMG-R). To begin molecular studies on pheromone production, a 1.16-kb complementary DNA representing approximately one-third of I. paraconfusus HMG-R was isolated by polymerase chain reaction and sequenced. The predicted translation product is 59% and 75% identical to the corresponding portion of HMG-R from the fruit fly, Drosophila melanogaster, and the German cockroach, Blattella germanica, respectively. Northern blots show that topical application of JH III increases HMG-R transcript levels in male thoraces in an apparent dose- and time-dependent manner. These data support the model that JH III raises HMG-R transcript levels, resulting in increased activity of the isoprenoid pathway and de novo pheromone production.

Insect tissues, not microorganisms, produce linoleic acid in the house cricket and the American cockroach.

Biosynthesis of linoleic acid, 18:2 (n-6), was unambiguously demonstrated to occur in the cockroach, Periplaneta americana, and the cricket, Acheta domesticus. Axenic tissue from both of these insect species was demonstrated by radio-gas-liquid chromatography (radio-GLC) and radio-high-performance liquid chromatography (radio-HPLC) to incorporate [1-14C]acetate and [1-14C]oleate into this essential fatty acid.

Characterization of the delta 12 desaturase in the American cockroach, Periplaneta americana: the nature of the substrate.

The cockroach, Periplaneta americana, can convert oleic acid (18:1(n - 9], to linoleic acid, (18:2(n - 6], by a microsomal delta 12 desaturase. Most of the desaturase activity was present in the fat body tissue, with lower activity in the epidermis and no detectable activity in the thorax or gut tissue. In incubations of microsomal preparations from fat body tissues with [1-14C]18:1-CoA, increased amounts of [1-14C]18:2 were found with increasing time and protein concentration. The form of the substrate for the delta 12 desaturase was determined to be 18:1-CoA by comparing activity towards [1-14C]18:1-CoA and [1-14C]18:1 transesterified to phospholipid. Ozonolysis of the 18:2 formed from [1-14C]oleoyl-CoA followed by radio-gas-liquid chromatography gave one labeled peak, 9-oxononanoate, which showed that the product of the delta 12 desaturase is the physiologically important isomer, 18:2(n - 6).

Mutations that affect circadian rhythms in Neurospora crassa can alter the reduction of cytochromes by blue light.

We have examined membrane fractions from mutant strains of Neurospora crassa that have altered responses to blue light or have altered circadian rhythms. Using an in vitro assay, we assessed whether the mutations affected the levels of photoreducible cytochromes. Three of the mutant strains, prd-1, rib-1, and wc-1, were not qualitatively different from the wild type. The poky strain was found to have high concentrations of photoreducible cytochrome c. After removal of this cytochrome, however, the photoreducible cytochromes in the plasma membrane and endoplasmic reticulum were also similar to those of the wild type. The most significant differences were found in strains mutated at the frq locus, which affects circadian rhythms. In the frq-9 strain, the cytochrome in the endoplasmic reticulum was not detectably reduced by blue light. The frq-1 mutation caused a significant shift in the spectrum of blue-light-reduced cytochrome in the endoplasmic reticulum.

Effects of dietary fish oil on human mammary carcinoma and on lipid-metabolizing enzymes.

The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic "nude" mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil- and corn oil-fed animals, and increased levels of 20:5n-3 and 22:6n-3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n-3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18:3n-3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The delta 9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the delta 6 and delta 5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The delta 5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the delta 5 and delta 6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.

Blue Light-Reducible Cytochromes in Membrane Fractions from Neurospora crassa.

We have assayed absorbance changes generated by blue light in plasma membranes, endoplasmic reticulum, and mitochondrial membranes from Neurospora crassa. Light minus dark difference spectra, obtained anaerobically in the presence of ethylenediaminetetraacetate, indicated that b-type cytochromes could be photoreduced in all three membranes. In plasma membranes, a b-type cytochrome with a distinct difference spectrum was photoreducible without addition of exogenous flavin. Addition of riboflavin greatly stimulated the photoreduction of cytochromes in endoplasmic reticulum and mitochondrial membranes. In its spectral characteristics the cytochrome on the endoplasmic reticulum resembled cytochrome b(5) or nitrate reductase, while the cytochrome in mitochondrial membranes had the same spectrum as cytochrome b of the mitochondrial respiratory chain.Cytochromes in the three membrane fractions reacted differently to blue light in the presence of various inhibitors. Potassium azide inhibited reduction of plasma membrane cytochrome b, with 50% inhibition at 1.0 millimolar. The same concentration of azide stimulated photoreduction of cytochromes in both endoplasmic reticulum and mitochondria. Although photoreduction of cytochromes in all three membranes was inhibited by salicylhydroxamic acid, cytochromes in plasma membranes were more sensitive to this inhibitor than those in endoplasmic reticulum and mitochondria. Cells grown to induce nitrate reductase activity showed an elevated amount of blue light-reducible cytochrome b in the endoplasmic reticulum.

Isolation and characterization of the Neurospora crassa endoplasmic reticulum.

The endoplasmic reticulum from Neurospora crassa was identified by monitoring the activity of the putative enzyme marker phosphatidylcholine glyceride transferase. After differential centrifugation of a cell homogenate, phosphatidylcholine glyceride transferase activity initially copurified with plasma membrane H+-ATPase. However, isopycnic centrifugation of the whole-cell homogenate on a linear sucrose gradient separated the two enzyme activities into different fractions. The lighter membrane fraction exhibited characteristics that have been associated with the endoplasmic reticulum in other organisms: (i) the inclusion of magnesium caused this light membrane fraction to shift to a higher density on the gradient; (ii) it was highly enriched in cytochrome c reductase, an endoplasmic reticulum marker in other systems; and (iii) the morphology of the light fraction with and without added magnesium was clearly distinguishable from that of the plasma membrane fraction by electron microscopy. A reinvestigation of the location of chitin synthetase confirmed its association with the plasma membrane fraction even after separation of the lighter fractions.